Rapid development of contrast agents in the recent years has generated a number of different formulations, which are useful in contrast-enhanced imaging of organs and tissue of human or animal body.
More recently, attention has been given to so-called “molecular imaging”, where suitable target specific components are used in the formulation of the contrast agents, for allowing selective contrast-enhanced imaging of organs or tissues. In addition, therapeutic use of contrast agent formulations, optionally in combination with molecular imaging, has also been described.
A class of contrast agents, particularly useful for ultrasound contrast imaging, includes suspensions of gas bubbles of nano- and/or micro-metric size dispersed in an aqueous medium. Of particular interest are those formulations where the gas bubbles are stabilized, for example by using emulsifiers, oils, thickeners or sugars, or by entrapping or encapsulating the gas or a precursor thereof in a variety of systems. These stabilized gas bubbles are generally referred to in the art with various terminologies, depending typically from the stabilizing material employed for their preparation; these terms include, for instance, “microspheres”, “microbubbles”, “microcapsules” or “microballoons”. The term “gas-filled microvesicles”, or shortly “microvesicles”, as used herein includes any of the above terminology.
The formulations of gas-filled microvesicles can be suitably modified, either for improving the diagnostic effect (e.g. through molecular imaging) and/or for therapeutic purposes, such as drug delivery and/or thrombolysis. For instance, microvesicles can be associated (e.g. by inclusion in their boundary envelope) with therapeutic agents and/or with specific components which are capable to link to a determined target within a patient's body (known as “targeting ligands”). Examples of targeting ligands include, for instance, peptides, proteins, antibodies, aptamers or carbohydrates capable of binding to specific receptors expressed by organs or tissues during pathogenic processes such as, for instance, angiogenesis, inflammation or thrombus formation.
Selectins (in particular P-, L- and E-selectin) are cell adhesion molecules expressed, among others, by vascular endothelium during inflammation processes. Selectin ligands and, in particular, P-selectin glycoprotein ligand-1 (PSGL-1: GenBank Acc. N° Q14242.1), is expressed constitutively on all leukocytes (neutrophiles, monocytes and most lymphocytes) and myeloid cells. As such, it plays a critical role in the tethering of these cells to activated platelets or endothelia expressing P-selectin and, even though with a lower affinity, to E and L-selectin. Examples of P-Selectin ligands are disclosed for instance in U.S. Pat. No. 5,840,679
International Patent Application Publ. No. WO 2008/131217 discloses microbubble compositions comprising targeting ligands directed to P-selectin. In particular, the targeting ligand is a fusion protein comprising a P-selectin ligand and a dimerization domain. In practical embodiments, the Application discloses the use of recombinant P-selectin ligand composed of the amino terminal region of PSGL-1 in a selectin-binding glycoform fused to the Fc portion of human IgG1 (rPSGL-Ig), to be conjugated via biotin-streptavidin binding to biotin containing microbubbles. While said Application does not disclose any exact sequence of the P-Selecting ligand, it refers to examples of P-selectin ligands and fragments thereof disclosed by US Patent Application Publ. No. 2003/0166521.
US 2003/0166521 discloses a PSGL-1 fusion protein (dimPSGL-1), also referred to as recombinant PSGL-Ig (or rPSGL-Ig), produced by truncating the N-term 47 amino acids of mature PSGL-1 and linking said N-term 47 amino acids of PSGL-1 to a Fc portion of human immunoglobulin G-1 (IgG1).
The Applicant has now observed that microvesicles bearing only a fragment of said rPSGL-1 protein may have some advantages when compared with microvesicles bearing the complete protein, for instance in terms of binding efficacy and/or in terms of stability of an aqueous suspension containing the microvesicles.